Isolation and reconstitution of the intestinal Na+/glucose cotransporter.
The intestinal Na+/glucose cotransporter was isolated from brush border membrane vesicles using a three-step procedure and Na(+)-dependent phlorizin binding as the measure of cotransporter enrichment. The initial step was to treat the Ca2(+)-precipitated brush border membrane vesicles with 0.02% sodium dodecyl sulfate (SDS) followed by sucrose gradient centrifugation which resulted in a 5-fold enrichment of the Na+/glucose cotransporter. The second step was chromatofocusing chromatography over the pH range from pH 7.4 to pH 4.0. This step resulted in an additional 20-fold purification as compared with the SDS-brush border membrane vesicle protein which served as the starting material. The final step was affinity chromatography on con A-Sepharose which resulted in a 5-fold enrichment of the chromatofocused protein. The glycoprotein fraction from the concanavalin A column reconstituted into phosphatidyl choline: cholesterol liposomes demonstrated Na(+)-dependent, phlorizin-sensitive, and osmotic strength-sensitive glucose uptake. This fraction consisted of a single 75-kDa polypeptide on SDS-polyacrylamide gel electrophoresis upon staining with silver. On the basis of these criteria it appears that a protocol for the isolation of the Na+/glucose cotransporter has been developed.
Peerce
B E
BE
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
Clarke
R D
RD
D000818:Animals; D002458:Cell Fractionation; D002784:Cholesterol; D002846:Chromatography, Affinity; D005947:Glucose / Q000378:metabolism*; D006023:Glycoproteins / Q000302:isolation & purification; D007413:Intestinal Mucosa / Q000032:analysis*; D008081:Liposomes; D008871:Microvilli / Q000032:analysis; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification* / Q000378:metabolism; D009994:Osmolar Concentration; D010713:Phosphatidylcholines; D012964:Sodium / Q000502:physiology
D006023:Glycoproteins; D008081:Liposomes; D009004:Monosaccharide Transport Proteins; D010713:Phosphatidylcholines; D002784:Cholesterol; D012964:Sodium; D005947:Glucose